Genetic Polymorphisms of GSTM1 and GSTT1 Genes in Delhi and Comparison with other Indian and Global Populations

All organisms are constantly and unavoidably exposed to a large number of foreign chemicals or xenobiotics. Most of these chemical carcinogens are not capable of inducing genetic damage themselves but require metabolic activation to electrophilic proximate carcinogens. Whether a compound contributes to cancer or other disease depends not only on the extent of an individual’s exposure, but also on the effectiveness of the individual’s ability to remove toxins from the body, involving phase-I (cytochrome P450) and phase-II (glutathione Stransferase) enzymes (Mannervik et al., 1992). Glutathione S-transferases (GSTs), a multigene family of phase-II metabolic enzymes, are active in the detoxification of a wide variety of potentially toxic and carcinogenic electrophiles by conjugating them to glutathione (Pemble et al. 1994). In mammals the eight classes of GSTs, i.e. alpha (GSTA), mu (GSTM), theta (GSTT), Pi (GSTP), zeta (GSTZ), sigma (GSTS), kappa (GSTK), and omega (GSTO) have been identified (Mannervik et al., 1992), based on sequence homology and substrate specificity. Among them GSTM1andGSTT1 polymorphisms are extensively studied. GSTM1 is situated in the GSTμ cluster, which is localised to chromosome 1 in region 1p13.3 and is involved in the detoxification of


Introduction
All organisms are constantly and unavoidably exposed to a large number of foreign chemicals or xenobiotics. Most of these chemical carcinogens are not capable of inducing genetic damage themselves but require metabolic activation to electrophilic proximate carcinogens. Whether a compound contributes to cancer or other disease depends not only on the extent of an individual's exposure, but also on the effectiveness of the individual's ability to remove toxins from the body, involving phase-I (cytochrome P450) and phase-II (glutathione S-transferase) enzymes (Mannervik et al., 1992).
Glutathione S-transferases (GSTs), a multigene family of phase-II metabolic enzymes, are active in the detoxification of a wide variety of potentially toxic and carcinogenic electrophiles by conjugating them to glutathione (Pemble et al. 1994). In mammals the eight classes of GSTs, i.e. alpha (GSTA), mu (GSTM), theta (GSTT), Pi (GSTP), zeta (GSTZ), sigma (GSTS), kappa (GSTK), and omega (GSTO) have been identified (Mannervik et al., 1992), based on sequence homology and substrate specificity. Among them GSTM1andGSTT1 polymorphisms are extensively studied. GSTM1 is situated in the GSTµ cluster, which is localised to chromosome 1 in region 1p13.3 and is involved in the detoxification of 1 polycyclic aromatic hydrocarbons and other mutagens. GSTT1 gene is located on chromosome 22q11.2 and is involved in the metabolism of small compounds found in tobacco smoke like mono halo methanes and ethylene oxide (Hayes and Pulford, 1995). The polymorphism in GSTM1 and GSTT1 gene loci is caused by a gene deletion which results in the absence of enzyme activity in individuals with the GSTT1 and GSTM1 null genotypes. These homozygous null polymorphisms of GSTM1 and GSTT1 may lead to wide inter-individual variations in the metabolic activation of chemical carcinogen (Board, 1981). The polymorphisms of GSTM1, GSTT1 have been associated with cancers of the lung, bladder, breast and colon (Autrup, 2000). Therefore, we evaluated the distribution of GSTM1, GSTT1 genotypes in Delhi population and compared it with GST polymorphism frequency in different states of India and with various populations worldwide.

Selection of controls
Peripheral blood from the controls was collected in vials containing ethlenediaminetetraacetic acid (EDTA) after receiving their informed consent. The controls selected for the study were either normal volunteers from the Institute, or normal healthy individuals visiting with the patients in various hospitals of Delhi, and were selected by social workers from our Institute who used to visit six major hospitals of Delhi for collection of samples from cervical cancer patients. Healthy individuals without any history of cancer were included in the study. Information on age, sex, smoking and alcohol habits was obtained. The ethical clearance was taken from our Institute.
GSTM1 and GSTT1 genotypes were determined by multiplex PCR using three sets of primers to amplify fragments of 218, 460 and 350bp for GSTM1, GSTT1 and Albumin gene (internal control) respectively (Arand et al., 1996) with slight modifications (Sharma et al., 2004). The primers used were GSTM1-forward 5'-GAA CTC CCT GAA AAG CTA AAG C-3'; and Albumin primers at 0.2µM each and 1.5 units of DNA AmpliTaq polymerase (Applied Biosystems) in a Perkin-Elmer thermal cycler. After an initial denaturation at 95°C for 5 min, amplification was carried out for 35 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min, followed by final extension at 72°C for 7 min. The products of multiplex PCR were separated by electrophoresis with ethidium bromide stained 3% agarose gel. Presence of amplicons of 218 bp, 460bp and 350bp revealed the presence of GSTM1, GSTT1 and Albumin (internal control) respectively ( Figure 1).

Statistical analysis
The data were tabulated and analysed. The mean±SD were estimated for quantitative data. T test was used for the comparison of age. The chi-square test was used to compare demographic data between various variables of the present study. The studied population was compared with reported frequencies from other neighbouring state populations of India and global populations; Europeans, Blacks and Asians.

Discussion
Homozygous deletions of GSTM1 or GSTT1 genes have an impaired ability to metabolically eliminate carcinogenic compounds and may therefore put such individuals at increased risk for various diseases. In the present study we have examined the polymorphism of GSTM1, GSTT1 genes in normal Delhi population.
There are substantial differences in the baseline frequencies of null genotypes for GSTM1 and GSTT1 in different ethnic groups. We have observed 33.6% individuals with GSTM1 homozygous null genotype in Delhi population which is comparable with data reported from different regions of India (Table 2) except in Trivandrum (17.0%) and Chennai (15.0 %), where the frequency is very low (Nair et al., 1999;Vijayalakshmi et al., 2005) respectively. Higher frequency of GSTM1 null genotypes was reported in two studies (49.2% and 59.9%) from Mumbai (Buch et al., 2002;Anantharaman, 2007). Otherwise, the frequency of GSTM1 null genotype was almost similar throughout India. We observed 12.4% of North Indians were homozygous null for the GSTT1 gene. The prevalence of GSTT1 homozygous null genotype is less (8.0-22.0%) in Indian population as compared to GSTM1 null (15.0-59.9%). The prevalence of GSTM1 homozygous null genotype is significantly higher in Caucasians (range: 41.8-53.5) and Asians (range: 49.0-65.2) as compared to Indian (range: 15.0-59.9) populations. The frequency of GSTT1 homozygous null genotypes is also significantly higher in blacks (range: 14.0-57.0) and Asians (16.0-84.6) ( Table 3). Very few results are available for the combined homozygous null genotypes of GSTM1and GSTT1. In the present study complete deletion of both GSTM1 and GSTT1 genotypes is observed in 10.8% individuals and the range in Indian population was between 4.5%-10.8%, whereas Nair et al. (1999) did not find any subject with homozygous null genotype for both GSTM1 and GSTT1 from Trivandrum. In Brazilian population, the range of both GSTM1 and GSTT1 homozygous null genotypes is between 4.5-17.0% and the frequency is higher in Asian population (19.6-37.0%) except Pakistanis and Indians from Singapore, where the frequency was low (5.0%) (Rehan et al., 2010). The high frequencies of GSTM1 and GSTT1 homozygous null genotypes observed in Chinese population are associated with the high incidence of oesophageal cancer. There were no significant differences between males and females in the frequency of GSTM1 or GSTT1 null genotypes in the present study. Similar results were reported in white and non-white individuals (Rossini et al., 2002).
Further, we have compared the frequency of GSTM1 and GSTT1 homozygous null genotype in three main world populations namely Blacks, Caucasians, Asians and compared with Indians (Table 3). These results were obtained by clubbing the individual data from the studies mentioned in Table 3 for all ethnic groups. GSTM1 and GSTT1 homozygous Null genotype was 31.9%; 24.9% in Blacks (N-3008), 32.4%; 16.2% in Indians (N=4509), 47.7%; 16.7% in Caucasians (N=2674) and 54.2%; 41.0% in Asians (N=4735) respectively. Considerable variations were observed in the frequencies of the homozygous null genotypes at the GSTM1 and GSTT1 loci among these four different ethnic groups. It shows that GSTM1 homozygous null genotype is lowest in Blacks, followed by Indians, Caucasians and Asians. At a glance these figures indicate that null alleles are higher in Caucasians and Asians (including Japanese, Chinese and Koreans). Cancer incidence is also higher in these populations specially the cancer of Lung, Colorectal, stomach, oesophagus and the Ovary (Parkin et al., 2005). Though not supported statistically here, it gives an indication that these two homologous null genotypes can account for racial differences in incidence of some important cancers in world populations. This may be in part due to their differing evolutionary histories and in part to differential selection arising from differing exposures to toxic substances, such as diet and tobacco and alcohol consumption.
This type of study would provide us the basic data for epidemiological studies. Therefore, GSTM1, GSTT1 polymorphism in combination with other detoxifying enzyme polymorphisms, could be used to identify highrisk individuals in clinical surveillance programmes. Individuals with different combinations of these alleles would also help in studying the effect of various carcinogens in different populations having various exposures. This data will be useful in designing various studies involving polymorphisms of GSTM1, GSTT1 genes and to compare results from various geographical regions of India.